Figure 7

hnRNP E1 overrexpression selectively affects spliced HIV-1 env RNA levels. (A) Shown is a diagram of the pgTat vector with the line below indicating the position of the probe (spanning both the 3'ss and polyadenylation sequence) used for RPA analysis. 293T cells were transfected with hnRNP E expressing plasmids as indicated, alongside pgTat and SVH6Rev. RNA was isolated 48 hours post transfection and used in RNase protection assays. Protection products were resolved on denaturing PAGE gels. Analysis of 3' end processing of unspliced env RNA by RNase protection assay. Identities of the various bands observed are indicated; unspliced, uncleaved (US/UC); unspliced, cleaved (US/C); spliced, uncleaved(S/UC); spliced, cleaved (S/C). (B) Graphical representation of the effect of hnRNP E proteins on the ratio of s/c to us/uc RNA. Averages of a minimum of three independent experiments are shown. Asterisk denotes values that were deemed significantly different from control at p < 0.05 (C) 293T cells were transfected with hnRNP E expressing plasmids as indicated, alongside Gag/RRE and SVH6Rev. 48 hrs post transfection RNA was isolated and used in RNase protection assays. Analysis of rev RNA levels by RNase protection assay. rev RNA abundance was corrected using the cotransfected VA control (pSPVA) and normalized to the vector control indicated by (-). (D) Summary of the effect of N- and C-terminal KH domain mutants of hnRNP E on rev RNA levels. Averages of a minimum of three independent experiments are shown. Asterisk denotes values that were deemed significantly different from control at p < 0.05